RNA editing is a novel and essential gene regulatory mechanism in trypanosomes that alters mitochondrial RNAs by specific uridine insertion and deletion to form functional mRNAs. The genetic information for editing is transferred from frans-acting gRNAs to mRNAs through base-pairing interactions. While considerable progress has been made in understanding the structure and composition of the core editing machinery, regulatory factors that affect the accuracy and efficiency of the editing process remain elusive. Our laboratory recently provided genetic evidence for the first RNA editing regulatory factor, the RNA binding protein, RBP16. RNAi-mediated knock-down of RBP16 in procyclic form (PF) trypanosomes leads to a dramatic and specific decrease in editing of apocytochrome b (CYb) RNA. CYb gRNA abundance is unaffected in RBP16 knock-downs, suggesting that regulation takes place at the level of gRNA utilization. Consistent with its role in vivo, RBP16 reproducibly stimulates RNA editing in vitro up to 5-fold. Preliminary data suggest that RBP16 enhances editing by an intriguing two-step mechanism, one aspect of which is independent of specific, high affinity RBP16-RNA interactions. Our goal is to determine the scope and mechanism of RBP16 RNA editing regulation. In Aim 1, we will down-regulate RBP16 expression in bloodstream form (BF) trypanosomes and analyze the effects on BF-specific and constitutive editing events. We will test the hypothesis that RBP16 facilitates recruitment of specific gRNAs to editosomes by analysis of editosome-associated gRNAs in PF and BF RBP16 knock-down cells. In Aim 2, we will directly test the model that RBP16 stimulates both pre- and post-mRNA cleavage steps of editing. The role the RNP1 and RGG RNA binding domains of RBP16 in this process will be examined by comparison of mutant and wild type RBP16 in the in vitro assay. In Aim 3, RBP16-interacting proteins will be identified by both expression of TAP-tagged RBP16 in PF and BF T. brucei and by a yeast two-hybrid screen. This combined genetic and biochemical approach will provide important insight into the largely unexplored area of RNA editing regulation.